Neurotransmitter release is one of the most regulated membrane fusion events. Unlike constitutive vesicle trafficking, synaptic vesicles are recruited to the presynaptic membrane, but do not readily fuse. Instead, an average of ten vesicles are stably docked at a region of the synapse termed the active zone awaiting an action potential. Synaptic vesicle fusion is closely associated with the Ca2+ influx that follows arrival of an action potential. Exocytosis is triggered within approximately 0.2 msec of the Ca2+ arrival. Although many key factors have been found to be essential for Ca2+-dependent neurotransmitter release, such as SNAREs, synaptotagmin, complexin, Munc18, and Munc13, the molecular mechanism of synaptic vesicle membrane fusion and Ca2+- triggering remains unclear. In this renewal application we wish to continue our efforts to decipher the molecular mechanism by using a combination of single molecule and single particle fluorescence imaging microscopy experiments. Driven by our hypothesis that SNAREs and auxiliary proteins do not fully assemble and, thus, do not trigger fusion until a Ca2+ signal arrives, we have designed labeling strategies to monitor distinct conformational states of the protein and protein-complexes involved in the process. We have also developed fluorescent reporters that provide information about content and lipid exchange as the system proceeds from vesicle docking, to membrane contact, and, finally, to fusion. These labeling strategies and the single molecule and single vesicle experimental setups are the cornerstones for the proposed research. Our assays are a breakthrough, since for the first time, we observe fast fusion kinetics upon Ca2+ triggering. Our new assay now enables us to determine the role of the factors involved, their stoichiometry (e.g., how many SNARE complexes and synaptotagmin molecules are required for fast Ca2+ triggered fusion?), to propose and test mutants of these factors that affect their function in the assay, and then to use these mutants as tools for experiments with neuronal cultures to dissect their role in vivo. Furthermore, we have recently demonstrated the ability of super-resolution imaging of synaptic proteins by using labeled antibodies to an unprecedented degree of localization accuracy. This in vivo imaging approach will enable us to image the distribution and correlation of synaptic proteins along axons, and so it will complement our reconstitution and single molecule biophysical studies.